Form Of Glyceraldehyde-3-phosphate Dehydrogenase Research Articles

Exposure of a specific pleioform of multifunctional glyceraldehyde 3-phosphate dehydrogenase initiates CD14-dependent clearance of apoptotic cells

Rapid clearance of apoptotic cells by phagocytes is crucial for organogenesis, tissue homeostasis, and resolution of inflammation. This process is initiated by surface exposure of various ‘eat me’ ligands. Though phosphatidylserine (PS) is the best recognized general recognition ligand till date, recent studies have shown that PS by itself is not sufficient for clearance of apoptotic cells. In this study, we have identified a specific pleioform of GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) that functions as an ‘eat me’ signal on apoptotic cell surface. This specific form of GAPDH which is exposed on surface of apoptotic cells was found to interact with CD14 present on plasma membrane of phagocytes leading to their engulfment. This is the first study demonstrating the novel interaction between multifunctional GAPDH and the phagocytic receptor CD14 resulting in apoptotic cell clearance (efferocytosis).

GAPDH: the missing link between glycolysis and mitochondrial oxidative phosphorylation?

The main function of glycolysis and oxidative phosphorylation is to produce cellular energy in the form of ATP. In the present paper we propose a link between both of these energy-regulatory processes in the form of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and CytOx (cytochrome c oxidase). GAPDH is the sixth enzyme of glycolysis, whereas CytOx is the fourth complex of the mitochondrial oxidative phosphorylation system. In MS analysis, GAPDH was found to be associated with a BN-PAGE (blue native PAGE)-isolated complex of CytOx from bovine heart tissue homogenates. Both GAPDH and CytOx are highly regulated under normal energy metabolic conditions, but both of these enzymes are highly deregulated in the presence of oxidative stress. The interaction of GAPDH with CytOx could be the point of interest as it has already been shown that GAPDH protein damage results in a marked decrease in cellular ATP levels. On the other hand, decreasing the ATP/ADP ratio may ultimately result in switching off the allosteric ATP inhibition of CytOx leading to increased ROS (reactive oxygen species), cytochrome c release and apoptosis. Moreover, we have previously reported that allosteric ATP inhibition of CytOx is responsible for keeping the membrane potential at low healthy values, thus avoiding the production of ROS and this allosteric ATP inhibition is switched on at a high ATP/ADP ratio. So, in the present paper, we propose a scheme that could prove to be a link between these two enzymes and their role in the prevalence of diseases.

The mTOR pathway senses the glucose sufficiency through an interaction between Rheb and Glyceraldehyde‐3‐phosphate dehydrogenase

Rheb (Ras homology enriched in brain) is a member of the Ras family, which acts as a positive regulator of mTOR signaling which controls the cell growth and cell cycle progression. We identify the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a binding partner of Rheb. GAPDH binds to Rheb directly and is co-immunoprecipitated with Rheb in cells overexpressing Rheb and GAPDH. Binding of Rheb to GAPDH is independent of Rheb nucleotide charging both in vivo and in vitro. We show that GAPDH substrate, glyceraldehyde-3-phosphate (G3P) inhibits the GAPDH-Rheb binding more effectively than the other factors do. The inhibition of glycolytic flux via glucose starvation or treatment of 2-deoxyglucose enhances the interaction between Rheb and GAPDH. We also find that glucose starvation inhibits the binding of Rheb to mTOR. Moreover, we show that GAPDH inhibits the mTOR kinase activity, which can be overcome by addition of G3P in mTOR kinase assay. In this study, we suggest there is the novel mechanism that the mTOR pathway integrates glucose sufficiency through direct interaction between glycolytic enzyme GAPDH and Rheb. GAPDH reflects the degree of glucose sufficiency through being charged with G3P, so the uncharged GAPDH (the form of GAPDH under glucose starvation condition) can bind to Rheb and lead to dissociation of Rheb from the mTOR complex.

Comigration of Two Autophagosome-Associated Dehydrogenases on Two-Dimensional Polyacrylamide Gels

Immunoblotting of two-dimensional polyacrylamide gels (pI 3-10) revealed six cytosolic molecular forms of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat hepatocytes. Two of the four full-length (~37 kDa) forms exhibited some binding to sedimentable cellular elements (but not to mitochondria), whereas one full-length and two short (~35 kDa) forms selectively bound to the membranes of autophagosomes and lysosomes. Tryptic fingerprinting by matrix-asssisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) confirmed the identity of the major full-length forms as GAPDH, but attempts to identify the major short form consistently suggested that this spot represented a different enzyme, 3-a-hydroxysteroid dehydrogenase (3aHSD). Silver staining indicated that this 3aHSD form would selectively bind to autophagosomal and lysosomal membranes. Immunoblotting of more focused 2D gels (pI 6-9) with an antibody raised against 3aHSD demonstrated immunostaining of four 3aHSD forms with masses of about 35 kDa. Autophagosomal membrane preparations were highly and selectively enriched with respect to all of these 3aHSD forms. One of them comigrated with the major short form of GAPDH, accounting for the paradoxical mass spectrometric identification of 3aHSD from this spot. Proteomic analysis by a combination of immunological and mass spectrometric identification methods was thus capable of resolving two comigrating dehydrogenases selectively associated with autophagic organelles.

Glyceraldehyde-3-phosphate dehydrogenase, a glycolytic enzyme present in the periplasm of Aeromonas hydrophila.

This is the first report describing the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a protein associated with the cell envelope of a gram-negative bacterium (Aeromonas hydrophila). Dose-dependent GAPDH activity was detected in whole bacterial cells from exponentially growing cultures, indicating that an active form of GAPDH is located outside the plasma membrane. This activity represents roughly 10-20% of total cell activity, and it is not reduced by pretreatment of the cells with trypsin. Assays with soluble GAPDH indicate that the activity measured in intact cells does not originate by rebinding to intact cells of cytosolic enzyme released following cell lysis. GAPDH activity levels detected in intact cells varied during the growth phase. The relationship between GAPDH activity and cell culture density was not linear, showing this activity as a major peak in the late-logarithmic phase (A600 = 1.1-1.3), and a decrease when cells entered the stationary phase. The late exponential growing cells showed a GAPDH activity 3 to 4-fold higher than early growing or stationary cells. No activity was detected in culture supernatants. Enzymatic and Western-immunoblotting analysis of subcellular fractions (cytosol, whole and outer membranes, and periplasm) showed that GAPDH is located in the cytosol, as expected, and also in the periplasm. These results place the periplasmic GAPDH of A. hydrophila into the family of multifunctional microbial cell wall-associated GAPDHs which retain their catalytic activity.

The Inactivation of the Acyl Phosphatase Activity Catalyzed by the Sulfenic Acid Form of Glyceraldehyde 3-Phosphate Dehydrogenase by Dimedone and Olefins

Abstract Treatment of the sulfenic acid form of glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) with a 2-fold molar excess of dimedone over the concentration of enzyme subunit completely inactivates the acyl phosphatase reaction catalyzed by the oxidized enzyme. The dehydrogenase activity catalyzed by the reduced form of the enzyme is not reactivated when the dimedone-inactivated enzyme is treated with dithiothreitol. When the acyl phosphatase is inactivated by [14C]dimedone ∼1 µg atom of 14C is incorporated per µeq of oxidized enzyme subunit which is not removed by gel filtration on Sephadex G-25. A 14C-labeled peptide from a tryptic digest of the acyl phosphatase which was inactivated with [14C]dimedone has been isolated in pure form and has been subjected to sequence analysis. This analysis has shown that [14C]dimedone forms a thioether derivative of Cys-149 by reacting with the sulfenic acid at the active site of the acyl phosphatase. The dehydrogenase activity is unaffected by [14C]dimedone when reduced glyceraldehyde 3-phosphate dehydrogenase is treated with [14C]dimedone and 14C radioactivity is not incorporated into the reduced protein. The acyl phosphatase activity catalyzed by the sulfenic acid form of glyceraldehyde 3-phosphate dehydrogenase isolated from pig muscle is also inactivated by 25 mm 3-cyclohexene-1-carboxylate at pH 5.1 in the presence of 0.1 m (NH4)2SO4 and other salts. Treatment of the inactivated acyl phosphatase with dithiothreitol does not reactivate the dehydrogenase reaction catalyzed by the reduced form of the enzyme. This indicates that treatment of the sulfenic acid form of the enzyme with the olefin leads to the covalent modification of Cys-149, the catalytically essentially sulfhydryl group for the dehydrogenase. Treatment of reduced glyceraldehyde 3-phosphate dehydrogenase with 3-cyclohexene-1-carboxylate under identical conditions has no effect on the dehydrogenase activity. The acyl phosphatase is also inactivated by 5.0 mm dihydropyran at pH 7.6 and by 30 mm tetrahydrophthalimide at pH 6.0. Treatment of the acyl phosphatase inactivated by these reagents with dithiothreitol does not reactivate the dehydrogenase activity catalyzed by the reduced form of the enzyme. Under identical conditions used to inactivate the acyl phosphatase, these two olefins do not inactivate the dehydrogenase activity catalyzed by native glyceraldehyde 3-phosphate dehydrogenase when added to it directly. Amino acid sequence analysis of the tryptic peptide which contains Cys-149 after the acyl phosphatase was inactivated with tetrahydrophthalimide suggests that an adduct between the sulfenic acid at the active site of the acyl phosphatase and tetrahydrophthalimide is formed during the inactivation.

Multiple Forms of Flounder Muscle Glyceraldehyde 3-Phosphate Dehydrogenase: SUBUNIT COMPOSITION, PROPERTIES, AND TISSUE DISTRIBUTION OF THE FORMS

Abstract The major forms of glyceraldehyde 3-phosphate dehydrogenase have been isolated from the muscle and liver of the marine fish winter flounder, Pseudopleuronectes americanus. The isolation procedure is a two-step process utilizing ammonium sulfate fractionation and column isoelectric focusing. The three muscle forms have isoelectric points of 8.4, 8.2, and 7.9, respectively. Electrofocusing of the crude soluble fraction yields the same pattern as the purified preparation, and refocusing of each form produces one peak at its respective isoelectric point. The 70 to 90% salt fraction lacks the 8.2 form while the 60 to 90% fraction contains it. The isolated muscle forms are homogeneous after electrophoresis on polyacrylamide gels with the 8.4 and 7.9 enzymes being completely resolved upon concurrent electrophoresis. The subunits appear to be identical as judged by electrophoresis in 8 m urea and 0.1% sodium dodecyl sulfate gels with a molecular weight of 39,000. The kinetic properties (Km values) of the muscle forms are similar as are the pH optima. The forms did display significant differences in their requirement for sulfhydryl-reducing agents in the assay mixture, number of p-chloromercuribenzoate-reactive sulfhydryl groups, heat stability (50°), and bound NAD content. Incubation of a muscle preparation in the presence of 40 mm NAD gave rise to two additional forms with pI values of 7.8 and 7.6. Flounder liver glyceraldehyde 3-phosphate dehydrogenase was prepared by a procedure similar to that used for muscle. The multiple form pattern differs markedly from that of muscle with the five major forms having pI values of 7.1, 6.8, 6.6, 6.5, and 6.3. The results indicate that the multimeric enzyme glyceraldehyde 3-phosphate dehydrogenase, consisting of apparently identical subunits, exists as a mixture of multiple forms having distinct properties. The evidence presented supports the concept of distinct conformational forms of a multimeric enzyme composed of one subunit type.

Tissue-characteristic forms of glyceraldehyde 3-phosphate dehydrogenase.

Glyceraldehyde 3-phosphate dehydrogenase exists in two different forms in various human tissue preparations. One of them is exhibited, after starch-gel electrophoresis, by a rapidly migrating or ;fast' band and the other by a ;slow' band. The proportion of the total activity in each of the two forms is characteristic of the type of tissue. A particulate fraction, obtained after centrifugation of homogenates, inhibits the enzyme activity and tends to convert the slow band into a fast one. The conversion is reversible. The fast band can also be converted into the slow one by addition of NAD(+) or ADP, or by dialysis against saturated sodium chloride solution. Conversions occur with the purified enzyme as well as with crude homogenates. The relevance of these findings to previous investigations and to glycolytic control mechanisms are discussed.